Protein tyrosine kinases perform diverse functions ranging from stimulation of cell growth and differentiation to arrest of cell proliferation. They are either receptor tyrosine kinases (RTK) or intracellular tyrosine kinases. Inhibition of kinase activity is recognized as an effective way to control disease in humans.
The activation loop (A-loop) plays a key role in the activation of kinases. Many kinases switch on and off depending on the conformational state of the A-loop. The structural nature of these changes involves the phosphorylation of 1-3 residues in the A-loop, which, in turn, leads to the formation of salt bridges in the catalytic loop (C-loop) and the N-lobe. The N-lobe comprises residues from N1049 to M1160. Three important motifs are located in this lobe; the P-loop, the αC-helix and the Lys-Glu ionic pair. The precise alignment of which plays a critical role in the kinase catalytic activity. Other kinases do not require the phosphorylation of these A-loop residues to become active. It is thought that these kinases adopt a unique conformation and, as a result acidic residues in the A-loop, form a salt bridge with a conserved arginine residue in the C-loop. This allows access to the ATP binding pocket.
Upon tyrosine residue phosphorylation, the A-loop adopts a configuration optimized for substrate binding and catalysis. In many kinase structures resolved to date, the A-loop, in the phosphorylated form, adopts similar conformations to satisfy catalytic constraints and to provide a platform for substrate binding.
The activation loops in the unphosphorylated kinases exhibit a wide range of different conformations, which may explain their role in kinase activity regulation. In this unphosphorylated state, the A-loop can assume conformations ranging from fully open to completely closed (Huse, M. and Kuriyan, J. 2002. Cell 109:275-282). The open, autoinhibited, conformation has been observed in the crystal structures of fibroblast growth factor receptor (Mohammadi, M. et al. 1996. Cell, 86, 577-587) and c-Met (Wang, W. et al. 2006. Proc. Natl. Acad. Sci. USA 103:3563-3568). In this conformation of the fibroblast growth factor receptor, the A-loop is not suitable for substrate binding but does not obstruct the ATP binding site. In the closed, canonical, autoinhibited conformation, the activation loop folds as a pseudosubstrate, obstructing binding of both ATP and the peptide substrate.
The canonical, autoinhibited conformation has been detected in the unbound insulin receptor tyrosine kinase IRK, one of the most studied receptor tyrosine kinases (Hubbard, S. R. et al. 1994. Nature 372:746-754), c-Src kinase bound to the ATP analog AMP-PNP (Xu, W. et al. 1999. Molecular Cell 3:629-638), Hck bound to a small molecule inhibitor (Schindler, T. et al. 1999. Mol. Cell. 3: 639-648), FLT3 receptor tyrosine kinase (Griffith, J. et al. 2004. Molecular Cell 13:69-178), c-Abl bound to Imatinib (Schindler, T. et al. 2000. Science 289, 1938-1942; Nagar, B. et al. 2002. Cancer Research 62:4236-4243), c-Kit tyrosine kinase receptor unbound and bound to Gleevec (Mol, C. D. et al. 2004. The Journal of Biological Chemistry 279:31655-31663), and more recently cFMS (colony stimulating factor receptor-1) bound to small molecule inhibitors (Schubert, C. et al. 2007. J. Biol. Chem. 282:4094-4101).
The majority of kinase inhibitors is believed to be interacting with the protein in a region which binds ATP. The conformation of the kinase, when bound by the inhibitors, is frequently very similar to the one in which ATP is bound, i.e., the active conformation of the kinase.
Typically, kinase inhibitors binding to the ATP pocket take advantage of limited sequence variations in the nucleotide binding site as well as conformational differences between phosphorylated and unphosphorylated forms of kinases. While phosphorylated forms may adopt similar conformations in different kinases, unphosphorylated, inactive conformations of kinases show great variability. Knowledge of these distinct kinase conformations allow rational drug design of high affinity, specific compounds.
A body of knowledge has given rise to the concept of ATP-competitive ligands and the use of X-ray crystallography to aid in their design. In such approaches it has become established that the ligand is required to at least in part to mimic the binding of ATP in the active site. The notion of these ligands competing for access to this site and on binding place the A-loop in a catalytically inactive conformation has been demonstrated by X-ray studies for the insulin-like receptor IGF1, IRK, cFMS, c-Abl, c-Kit, Flt3, MusK, etc.
More recently, a different type of kinase inhibitor has become known in the field. These new inhibitors appear to interact with an inactive form of the kinase in the ATP binding site. Stabilization of unphosphorylated inactive forms of RTKs provides a different approach to modulate signaling through kinases. It provides another mechanism to control over expression and non-ligand activation.
Controlling the position of the activation loop by use of small molecule inhibitors has been documented and shown by the use of X-ray crystallography. This technique provides a detailed structural insight into the mechanism by which the A-loop is prevented from achieving a catalytically active conformation. It is this technique which provides important and valuable information for the use of the design of more efficacious and selective kinase inhibitors.
In the case of the ATP-competitive ligand STI-571 binding to the inactive form of c-Abl, X-ray studies show that DFG is in the out conformation, and that the A-loop is held in an inactive conformation (Nagar, B. et al. 2002. Cancer Research 62:4236-4243).
Despite the identification of many agents which have been described to affect such control there remains a need for additional, novel and selective agents which offer the benefits of increased specificity and reduced side effects. Despite many reports, there remains a need to identify methods to inhibit the signaling through this important class of proteins.
The references cited herein are not admitted to be prior art to the claimed invention.